Two new fatty acid derivatives from the stem bark of alchornea laxiflora (euphorbiaceae)

Article abstract of DOI:10.1007/s11746-011-1770-7

Euphorbiaceae is a family of plants used in traditional remedies in central Africa to treat selected diseases. Some of the phytochemical components in the stem bark of Alchornea laxiflora that have biochemical activity were identified. A number of novel compounds were isolated, including a new fatty acid ester, (1) a new ceramide, (2) some triterpenoids, (3-5), ellagic acid (6) and its derivatives (7, 8) were isolated. The structures of these compounds were determined on the basis of spectroscopic methods as well as HR-ESI-TOF-MS analysis, chemical transformation and by comparison of their physical and spectral data with those reported in the literature. The cytotoxicity of some isolated compounds was investigated against human promyelocytic leukaemia (HL60) cell line by using the MTT method. Compounds 1, 4 and 5 showed a cytotoxic activity with IC₅₀ at 58.7, 6.6 and 6.8 μM, respectively.

Full text of DOI:10.1007/s11746-011-1770-7

J Am Oil Chem Soc (2011) 88:1153–1159
DOI 10.1007/s11746-011-1770-7
ORIGINAL PAPER
Two New Fatty Acid Derivatives from the Stem Bark of Alchornea
laxiflora (Euphorbiaceae)
•
Louis Pergaud Sandjo Herve M. Poumale Poumale Xavier Noudou Siwe
•
•
´
Hippolyte Nga Ntede Yoshihito Shiono Bonaventure Tchaleu Ngadjui
•
•
•
•
•
Rui M. W. Krause Derek Tantoh Ndinteh Joseph Tanyi Mbafor
Received: 28 October 2010 / Revised: 12 January 2011 / Accepted: 17 January 2011 / Published online: 3 February 2011
Ó AOCS 2011
Abstract Euphorbiaceae is a family of plants used in
traditional remedies in central Africa to treat selected dis-
eases. Some of the phytochemical components in the stem
bark of Alchornea laxiflora that have biochemical activity
were identified. A number of novel compounds were iso-
lated, including a new fatty acid ester, (1) a new ceramide,
(2) some triterpenoids, (3–5), ellagic acid (6) and its
derivatives (7, 8) were isolated. The structures of these
compounds were determined on the basis of spectroscopic
methods as well as HR-ESI-TOF-MS analysis, chemical
transformation and by comparison of their physical and
spectral data with those reported in the literature. The
cytotoxicity of some isolated compounds was investigated
against human promyelocytic leukaemia (HL60) cell line
by using the MTT method. Compounds 1, 4 and 5 showed
a cytotoxic activity with IC50 at 58.7, 6.6 and 6.8 lM,
respectively.
Keywords Euphorbiaceae ꢀ Alchornea laxiflora ꢀ
Isolation ꢀ Fatty derivatives ꢀ Cytotoxicity
Abbreviations
CC
Column chromatography
COSY
ESI-MS
FT-IR
HMBC
HSQC
Correlation spectroscopy
Electrospray mass spectrometry
Fourier transformed infra-red
Heteronuclear multiple bond correlation
Heteronuclear single quantum correlation
L. P. Sandjo (&) ꢀ H. M. P. Poumale ꢀ X. N. Siwe (&) ꢀ
D. T. Ndinteh ꢀ J. T. Mbafor
HR-ESI-MS Higher resolution electrospray mass
spectrometry
´
Department of Organic Chemistry, University of Yaounde I,
´
P.O. Box 812, Yaounde, Cameroon
e-mail: plsandjo@yahoo.fr
m.p.
Melting point
NMR
NOESY
TLC
Nuclear magnetic resonance
Nuclear overhauser effect spectroscopy
Thin layer chromatography
Vacuum column chromatography
X. N. Siwe
e-mail: xaviersiwe@yahoo.fr
VCC
H. M. P. Poumale ꢀ Y. Shiono
Department of Bioresource Engineering, Faculty of Agriculture,
Yamagata University, Tsuruoka, Yamagata 997-8555, Japan
H. N. Ntede
National Advanced School of Engineering,
´ ´
University of Yaounde I, P.O. Box 8390, Yaounde, Cameroon
Introduction
B. T. Ngadjui
Department of Pharmacy and Traditional Pharmacopeia,
Faculty of Medicine and Biomedical Sciences, University
Alchornea laxiflora is a shrub of the Euphorbiaceae
family which is common throughout central, eastern and
southern Africa. Plant material used in this investigation
was harvested in Cameroon. The stems, especially the
small branches are used in Nigeria as chew-sticks and its
leaves are also used in this country to preserve kola nuts
[1]. The decoctions of the leaves of A. laxiflora are also
´ ´
of Yaounde I, P.O. Box. 8664, Yaounde, Cameroon
X. N. Siwe ꢀ R. M. W. Krause ꢀ D. T. Ndinteh
Department of Chemical Technology,
University of Johannesburg, P.O. Box 17011,
Doorfontein, Johannesburg 2028, South Africa
123

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J Am Oil Chem Soc (2011) 88:1153–1159
employed to treat inflammation and some infections [1].
Previous phytochemical studies carried out on this
species by Ogundipe et al. [2] showed the presence of
alkaloids, cardiac glycosides, saponins and phenolic
compounds with a higher concentration in the leaves than
in the roots. It seems that the biological effect usually
observed in folk medicine are induced by these second-
ary metabolites. So, the previous works [2, 3] and the
traditional uses have stimulated our interest in chemically
evaluating A. laxiflora and in the biology of the com-
pounds obtained.
3-O-acetyl of oleanolic acid 4 (9.5 mg); from the same
ratio of hexane/ethyl acetate 3 mg of 3-O-acetyl of ursolic
acid 5 were obtained. The fractions 61–68 eluted with
hexane/ethyl acetate in the ratio 4:1 (v/v) afforded 8 mg of
compound 1. From the fraction B eluted with methylene
chloride/methanol in gradient conditions, 165 fractions
were obtained and 7 mg of compound 2 were isolated from
the fractions 30–40 eluted in the ratio 97.5:2.5 (v/v); 4 mg
of 3-O-methylellagic acid 7 were further obtained from the
fractions 45–51 in the ratio 95:5 (v/v); from the same ration
2.3 mg of 3-O-b-D-glucopyranoside of b-sitosterol 3 were
filtered. From C eluted with methylene chloride/methanol
in the gradient condition, 2 mg of ellagic acid 6 were
isolated from the fractions 32–40 eluted in the ratio
92.5:7.5 (v/v) and from the fractions 50–58, 4.5 mg of
3-O-methyl-30-O-a-rhamnopyranosylellagic acid 8 were
collected from the ratio 9:1 (v/v).
We hereby report the isolation and structural elucidation
of the new compounds based on the spectrometric methods
and chemical transformation. Their cytotoxic activities will
be also presented.
Experimental Part
Vacuum column chromatography (VCC), column chro-
matography (CC) and thin layer chromatography (TLC)
were performed over silica gel 60H (particle size 90%
\45 mm), or 200–300 mesh silica gel silica gel GF254,
respectively. The melting point (m.p.) was measured by a
Stuart Scientific Melting Point apparatus SMP3; uncor-
rected. Optical rotations: Perkin Elmer polarimeter model
341 at 589 nm. IR Spectra: Perkin-Elmer FT-IR system
Methods
Methanolysis
Methanolysis of compound 2—(1 mg) was refluxed
(70 °C) for 18 h in 2.5 mL of MeOH containing 1.5 mL of
0.9 N HCl under magnetic stirring. The mixture was neu-
tralized with an aqueous solution of Na2CO3 and extracted
with CHCl3. The fatty acid methyl ester was carefully
characterized by ESI-MS at m/z 441 [C28H56O3?H]?.
1
spectrum BX spectrometer, KBr disks. H and 13C NMR:
Bruker DRX-400 MHz for 1D- and 2D-NMR spectra.
HR-ESI-MS and ESI-MS were recorded with a JEOL
JMS-700 instrument.
Acetylation and Oxidative Cleavage
of the Double Bond
Plant Material
This was carries out by taking 1 mg (3.18 lmol) of 1 and
1 mg (1.078 lmol) of 2 and these were separately dis-
solved in 1 mL of pyridine and 1.5 mL of acetic anhydride.
The reaction under magnetic stirring at room temperature
was stopped after 1 h and the usual work up gave the
acetylated products. 10 and 20 were separately dissolved in
4 mL of a mixture of dioxane/water (3:1); two equivalents
of pyridine, two drops of 4% by weight of an aqueous
solution of OsO4 and four equivalents of NaIO4 were
added. The reaction was left under magnetic stirring at
room temperature for 3 h. Quenching was done with
10 mL of an aqueous solution of sodium thiosulfate and
left under magnetic stirring during about 20 min. The
organic parts were extracted three times with 20 mL of
CH2Cl2 for each reaction and concentrated separately
under vacuum. The separate analyses by ESI-MS of dif-
ferent organic residual phases gave important peaks at
m/z 316 [C15H24O7?H]? and 249 [C15H30O?Na]? from 1
and 2, respectively.
´
A. laxiflora (Euphorbiaceae) was collected From Yaounde
central region of Cameroon in August 2006. A sample (No
45363HNC) has been deposited in the National Herbarium
´
of Yaounde, Cameroon.
Extraction and Isolation
The dried stem bark of A. laxiflora (2.7 kg) was cut into
small pieces, powdered and extracted by maceration with
10 L of a mixture of methylene chloride/methanol (1:4,
v/v) for 72 h. The organic extract was concentrated to yield
87 g of crude extract which was subjected to flash chro-
matography [SiO2, hexane/ethyl acetate (3:1, v/v), ethyl
acetate and ethyl acetate/methanol (9:1, v/v)] yielding three
fractions A, B and C. A was purified by CC with different
mixtures of hexane/ethyl acetate in order of increasing
polarity yielding 150 fractions. The ones (36–39) eluted
with hexane/ethyl acetate in the ratio 19:1, (v/v) yielded
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J Am Oil Chem Soc (2011) 88:1153–1159
Chemical Properties of Compounds
1155
(Ref. [5]); ESI-MS m/z 499.37 [C31H50O4?H]?; Some
characteristic H- and 13C-NMR data (CDCl3) dH/dC 4.72
1
(10Z)-Tetradec-10-enoic acid-(2S)-2-carboxy-2-
hydroxyethyl ester (1)
(H-3, dd, 2.5, 9.5 Hz)/80.9, 5.45 (H-12, brs)/122.6 (C-12),
143.6 (C-13), Acetyl group [a-CH3, 2.50 (s)/22.4; C=O
(171.0)],
Yellow viscous oil; [a] ? 4.6 (c 0.06, DMSO); IR 3,430,
2,930, 2,863, 1,686 and 1,644 cm-1; HRESIMS m/z:
313.2018 [C17H30O5–H]-, 239.1651 [C17H30O5–CO2–
3-O-Acetyl of Ursolic Acid (5)
1
C2H6–H]-, 183.1388 [C17H30O5–C3H4O3–C3H6–H]-; H
White powder; positive response in the Liebermann Bur-
chard test characteristic of triterpenes; m.p. [275–277] °C
(Ref. [4]); ESI-MS m/z 577.44 [C35H60O6?H]?; Some
and 13C NMR: Table 1.
1
Characteristic H- and 13C-NMR data (CDCl3) 4.49 (H-3,
(2R)-2-Hydroxy-N-[(2S,3S,4R,15Z)-1,3,4-trihydroxy-15-
triaconten-2-yl]octacosamide (2)
dd, 2.1, 10.1 Hz)/80.3, 5.25 (H-12, brs)/124.4 (C-12),
138.2 (C-13), acetyl group [a-CH3, 2.11 (s)/22.1; C=O
(170.8)].
White solid; m.p. 139.5–140.5; [a] - 8.05 (c 0.090, C5H5N);
IR: 3,335, 3,201, 1,649, 1,637 and 1,542 cm-1; HRESIMS
m/z: 928.8678 [C58H115NO5?Na]?, 878.8289 [M–CH3OH–
H2O?Na]?, 860.8191 [M–CH3OH–2H2O?Na]?, 850.4157
[M–CH3OH–H2O–C2H4?Na]?, 822.7675 [M–CH3OH–
H2O–2C2H4?Na]?; 1H and 13C NMR: Table 2.
Ellagic Acid (6)
Yellowish powder; dark green color with FeCl3, m.p.
358 °C (after decomposition) (Ref. [6]); m/z 303.01
[C14H608?H]?; 1H- and 13C-NMR data (DMSO-d6) dH
7.40 (2H, s, H-5 and H-50); dC 106.4 (C-1 and C-10), 108.9
(C-5 and C-50), 112.4 (C-6 and C-60), 135.8 (C-2 and C-20),
140.9 (C-3 and C-30), 148.3 (C-4 and C-40), 159.2 (C-7 and
C-70).
3-O-b-D-Glucopyranoside of b-Sitosterol (3)
White powder; Positive response in the Liebermann Burchard
and Molish tests characteristic of glycoside of steroid;
m.p. [290–291] °C (Ref. [4]); ESI-MS m/z 577.44
1
[C35H60O6?H]?; Some Characteristic H- and 13C-NMR
3-O-Methylellagic Acid (7)
data (C5D5N) dH/dC 5.35 (H-6, brs)/121.7 (C-6), 140.9 (C-5)];
b-D-glucopyranosyl: 101.2, 70.5, 73.9, 77.2, 77.2, and 61.5.
Yellowish powder; dark green color with FeCl3, m.p.
1
[302–303] °C (Ref. [7]); m/z 317.03 [C15H808?H]?; H-
and 13C-NMR data (DMSO-d6) dH 7.50 (1H, s, H-5), 7.44
(1H, s, H-50), 4.02 (3H, s, 3-OMe). dC 158.9 (C-7), 158.8
(C-70), 152.1 (C-4), 148.3 (C-40), 141.5 (C-2), 140.1 (C-3),
139.9 (C-30), 136.1 (C-20), 112.5 (C-10), 112.2 (C-6), 111.9
3-O-Acetyl of Oleanolic Acid (4)
White powder; Positive response in the Liebermann Bur-
chard test characteristic of triterpenes; m.p. [257–259] °C
1
Table 1 The H-NMR (400 MHz) and 13C-NMR (100 MHz) spectral data of compound 1 measured in CDCl3; d in ppm, J in Hz
Position
dH
dC (APT)
Cosy
HMBC
1
–
172.8
–
–
2
2.30 (t, 6.3)
1.60 (br s)
1.30 (br s)
2.00 (m)
34.2
1.60
24.8, 29.0–29.7, 172.8
29.0–29.7, 172.8
27.2, 24.8, 22.5, 29.0–29.7
29.0–29.7, 129.6, 130.1
27.2
3
24.8
1.30, 2.30
0.83
4–8, 13
22.5, 24.8, 27.1, 29.0–29.7
9
27.2
1.30, 5.36, 5.37
2.00
10
11
12
14
10
20
30
5.36 (m)
129.6
130.1
31.9
5.37 (m)
2.00
27.2
2.00 (m)
1.30
29.0–29.7, 129.6, 130.1
22.5, 29.0–29.7, 31.9
–
0.83 (t, 6.3)
–
14.1
1.30
173.2
68.8
–
5.22 (br t, 6.3)
4.15 (dd, 2.3, 11.4)
4.25 (dd, 6.4, 11.4)
4.15, 4.25
4.25, 5.22
4.15, 5.22
62.1, 173.2
62.1
68.8, 172.8, 173.2
68.8, 172.8, 173.2
123

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J Am Oil Chem Soc (2011) 88:1153–1159
1
Table 2 The H-NMR (400 MHz) and 13C-NMR (100 MHz) spectral data of compound 2 measured in C5D5N; d in ppm, J in Hz
Position
dH
dC (APT)
Cosy
HMBC
NH
8.62 (d, J = 8.0 Hz)
4.53 (m, Ha) 4.47 (m, Hb)
5.16 (m)
–
5.16
53.2, 175.5
53.2, 77.1
62.2, 77.1, 175.5
53.2
1
62.2
4.47, 4.53, 5.16
4.47, 4.53, 4.37, 8.62
–
2
53.2
3
4.37 (br d, 8.0 Hz)
4.32 (m)
77.1
4
73.1
2.01, 2.02, 4.37
1.27–1.33, 4.32
0.87, 2.02, 2.25
5.55
33.5, 53.2, 62.2
29.7–30.2, 73.1
14.5, 23.2
131.0
5
2.01 (m, Ha), 2.02 (m, Hb)
1.27–1.33 (br s)
2.02 (m)
33.5
6–13, 18–30, 50–280
22.5, 23.9–30.2, 31.9
14
27.2
130.9
131.0
32.0
14.5
175.5
72.7
35.9
24.8
15
5.54 (d, 4.0 Hz)
5.55 (d, 4.0 Hz)
2.20 (m)
2.02, 2.20
2.02, 2.20
5.54
32.0
16
32.0
17
30, 280 (CH3)
10 (C=O)
20
30
40
130.9
0.87 (t, 8.0 Hz)
–
1.27–1.33
–
23.2, 29.7–30.2
–
4.65 (m)
2.03, 2.25
1.27–1.33, 1.80
2.01
27.2, 35.9, 175.5
27.2, 72.7
35.9
2.03 (m, Ha), 2.25 (m, Hb)
1.80 (m, Ha)
2.01 (m, Hb)
1.80
(C-1), 111.3 (C-5), 110.2 (C-50), 107.2 (C-60),60.9
(3-OMe).
Results and Discussion
The crude methanol extract was subjected to repeated
column chromatographic separations identifying a new
fatty acid ester 1, a new ceramide 2,3-O-b-D-glucopyran-
oside of b-sitosterol 3 [8], 3-O-acetyl of oleanolic acid 4
[9], 3-O-acetyl of ursolic acid 5 [9], ellagic acid 6, 3-O-
methylellagic acid 7, and 3-O-methyl-30-O-a-rhamnopyr-
anosylellagic acid 8 [10].
3-O-Methyl-3’-O-a-rhamnopyranosylellagic acid (8)
Yellow gum, dark green color with FeCl3 and gave positive
response with Molish test characteristic of glycoside of
phenolic compound; m/z 463.09 [C21H18012?H]?; 1H- and
13C-NMR data (CD3OD) Aglycon: dH 7.46 (H-5, s), 7.48
(H-50, s), 4.11 (CH3–O), rhamnopyranosyl: 5.72 (1H, d,
1.6), 4.34 (1H, dd, 1.5, 3.4), 4.04, 3.44 (1H, t, 9.6), 4.45
(1H, m), 1.23 (3H, d, 6.2); Aglycon: dC 112.2, 142.4, 143.5,
154.4, 114.1, 113.3, 161.8, 110.6, 143.6, 140.3, 153.7,
115.2, 114.6, 161.8, 61.3. Rhamnopyranosyl: 102.7, 72.0,
72.2, 73.9, 71.7, 17.7
Compound 1 was isolated as a yellow viscous oil from
hexane/ethyl acetate (3:1, v/v) and had been detected at
m/z 313.2018 in the negative mode HR-ESI-TOF-MS. This
mass calculated at m/z 313.2020 corresponded to the
molecular formula [C17H30O5–H]- accounting for three
double bond equivalents. The analysis suggested a fat which
was supportedbythe1H-and 13C-NMRspectra[11]. A triplet
of 3H at dH/dC 0.83/14.1 (J = 6.3 Hz), a broad singlet of 6H
at dH/dC 1.30/29.0–29.7, a multiplet of 2H at dH/dC 1.60/24.8
which is CH2 group b position of C=O function, a CH2 group
in a position C=O function appearing as a triplet at dH/dC
2.30/34.2 and the carbonyl of ester function at dC 172.8 were
displayed. Besidestheseresonances, fourappeareddownfield
that consisted of one carbinol at dH/dC 4.15 (dd, J = 2.3,
11.4 Hz), 4.25 (dd, J = 6.4, 11.4 Hz)/62.1, an oxymethine at
dH/dC 5.22 (br t, J = 6.3 Hz)/68.8 and two olefinic protons at
dH/dC 5.36 (m)/129.6 and at dH/dC 5.37 (m)/130.1. Its con-
figuration is Z since the chemical shift of allylic protons were
revealed at dC 27.2 [12] and its position D10 was determined
after acetylation and oxidative cleavage reactions. The
product obtained was analysed by ESI-MS which produced a
Cell Viability
Human promyelocytic leukemia HL-60 (RCB-0041,
4 9 104 cells/mL) cells were treated with the compounds
at various concentrations at 37 °C under a humidified, 5%
CO2 atmosphere for 4 days in RPMI 1640 medium sup-
plemented with 10% fetal bovine serum, 50 units/mL of
penicillin and 50 lg/mL of streptomycin and cytotoxicity
was determined using a 96 plates with MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide]
assay. The percentages of viable cells were calculated as
ratio of the A570 values of treated and control cells (treated
with 2% MeOH). The values are the average of two
independent experiments.
123

J Am Oil Chem Soc (2011) 88:1153–1159
1157
Scheme 1 Oxidative cleavage
confirming the double bond
position in the fatty acyl moiety
of compound 1
O
O
10
O
HO
O
2
OAc
1) C5H5N/(CH3CO)2O (1:1)
Room Temp.
m/z 317 [C15H24O7+H]+
Compound 1
2) OsO4 (cat)-NaIO4 (4eq)
C5H5N (2eq);Dioxane/H2O (3:1)
Room Temp.
+
O
peak at m/z 317 corresponding to [C15H24O7?H]?
(Scheme 1). The HMBC and COSY spectra showed corre-
lations from the gem-protons at dH 4.15 and 4.25 and the
oxymethine signal at dH 5.22 to the carbonyl function at dC
173.2 and these information indicated that 1 is a glyceric acid
derivative. The downfield gem-proton signals at dH 4.15
and 4.25 corresponded to esterification with a fatty acyl
moiety and this was evident by the correlation further
observed on the HMBC spectrum between this carbinol and
another carbonyl function at dC 172.8. The absolute config-
uration at C-20 was established from the positive optical
rotation, which is a general feature of long-chain 1-O-alkyl-
sn-glycerols [13].
1.30/(29.7–30.2) corresponding to the sequence of CH2 group
was further observed as well as signals of H–N group at dH
8.62 (d, J = 8.0 Hz) and azamethine proton (HC–N) at dH/dC
5.16 (m)/53.2. Furthermore, the chemical shifts of a double
bond having cis configuration [10] were also observed at
dH/dC 5.54 (d, J = 4.0 Hz)/130.9 and 5.55 (d, J = 4.0 Hz)/
131.0 since the allylic methylene groups appeared between
26.5–32.0 ppm instead of a resonance more than 32.0 ppm in
the case of trans configuration. The foregoing data allowed us
to suggest that, 2 is a ceramide flanked with an olefinic
function. Some correlations ascertaining the amide function
were observed between the proton H–N at dH 8.62 and the
0
carbonyl at dC 175.5 as well as from the proton H2 at dH/dC
4.65 (br s)/72.7 to the carbonyl function (175.5) and the
0
0
successive CH2 groups [C3 (dC 35.9), C4 (dC 24.8)]. The
last observation confirmed that the fatty acid moiety is
a-hydroxylated. The methanolysis (1N hydrochloride
acid:methanol, at 70 °C for 18 h) of compound 2 gave the
fatty acid methyl ester and the long chain base which were
carefully characterized onthe baseofHRESI-MS. The peakat
m/z 441.4315 corresponding to the molecular formula
[C28H56O3?H]? showed that the fatty acid methyl ester has
one unsaturation attributed to the carbonyl function. The
position of the olefin function was evident by oxidative
cleavage after acetylation and the fatty aldehyde was further
identified by the mass spectrometry analysis which showed
thepeakatm/z249 [C15H30O?Na]? (Scheme 2). Therelative
configuration was defined with NOESY spectrum which
presented interactions between the signals at dH 5.16 (H-2),
4.32 (H-4) and the one at dH 4.53 (H-1a) while H-1b at dH 4.47
interacted with H-3 (dH 4.37). The absolute configuration at
C-2, C-3, C-4, and C-20 was determined to be (S), (S), (R), and
O
1'
O
1
14
3
2'
11
HO
O
3'
2
10
4
OH
From the foregoing data, compound 1 was identified as (2S)-
3-[(10Z)-tetradec-10-enoyloxy]-2-hydroxypropanoic acid.
Compound 2 was obtained as a white solid from methylene
chloride/methanol (39:1, v/v). Its positive mode HR-ESI-
TOF-MS spectrum showed a pseudo molecular ion at m/z
928.8678 (calc. 928.8667) corresponding to the molecular
formula [C58H115O5N?Na]? accounting for two double bond
equivalents. TheIRspectrumexhibitedanabsorptionbandfor
a free OH group (3,335 cm-1) [14], two strong absorption
bands for olefinic function (1,649 cm-1) and for secondary
amide (3,201, 1,637 and 1,542 cm-1). The NMR spectra of 2
showedsignalsclosetothoseofphytoceramides[14].Atriplet
of 6H at dH/dC 0.87, J = 8.0 Hz/14.5 assigned to the two
terminal CH3 groups was observed. A broad singlet at dH/dC
1
(R), respectively, by comparison of H- and 13C-NMR data
with those obtained from the literature [15].
2'
HO
28'
3'
1'
30
O
NH OH
3
HO
1
5
16
4
2
15
OH
123

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J Am Oil Chem Soc (2011) 88:1153–1159
Scheme 2 Oxidative cleavage
confirming the double bond
position in the amino alcohol
side of compound 2
AcO
C26H53
1) C5H5N/(CH3CO)2O (1:1)
Room Temp.
O
NH
OAc
Compound 2
AcO
2) OsO4 (cat)-NaIO4 (4eq)
C5H5N (2eq);Dioxane/H2O (3:1)
Room Temp.
OAc
+
O
O
m/Z 249 [C15H30O+Na]+
Table 3 Cytotoxic activity against human promyelocytic leukaemia
(HL60) cell line
The chemical study of these species led to the identification
of two new fatty acid derivatives together with two pen-
tacyclic triterpenes, one steroid glucoside, ellagic acid and
some ellagic acid derivatives. Beside the antimicrobial and
antioxidant activities [17, 18] reported in this plant, some
of isolated compounds have cytotoxic activity against a
human promyelocytic leukaemia (HL60) cell line. The
above-mentioned data indicate that A. laxiflora should be
submitted to a large biological screening since the results
could provide crucial information about the appropriate
dosage. This could also be helpful for traditional healers
working with phytomedicines.
Compounds
IC50 (lM)
Compound 1
58.7
[100
6.6
Compound 2
3-Acetyloleanolic acid 4
3-Acetoxyursolic acid 5
3-O-Methylellagic acid 7
3-O-Methylellagic acid-30-O-a-rhamnopyranoside 8
6.8
[100
[100
Camptothecin = 0.02 lM was used as positive control
Acknowledgments Dr. H. M. P. Poumale is thankful to the JSPS
(Japan Society for the Promotion of Science) for the postdoctoral
fellowship through their financial support (P08430) and research stay
at the University of Yamagata.
All the information above-mentioned led us to identify 2
as being (2R)-2-hydroxy-N-[(2S,3S,4R,15Z)-1,3,4-trihy-
droxy-15-triaconten-2-yl]octacosamide.
Cytotoxic Assay
References
The cytotoxicity of compounds 1, 2, 4, 5, 7 and 8 were
tested against HL-60 (human promyelocytic leukemia)
under in vitro conditions. Compounds 1, 4 and 5 showed a
cytotoxic activity and the IC50 are in Table 3. The penta-
cyclic triterpenoids (4 and 5) were more active than the
fatty acyl ester (1). However, only compounds having less
hydrophilic functions had cytotoxic activity. The lack of
D4,5 double bond in the ceramide structure could explain its
inactivity since the olefinic bond at this position is crucial
for apoptosis inducing activity. [16].
1. Olatunde FE (2003) African indigenous plants with chemother-
apeutic potentials and biotechnological approach to the produc-
tion of bioactive prophylactic agents. Afr
12:662–671
J Biotechnol
2. Ogundipe OO, Moody JO, Houghton PJ, Odelola HA (2001)
Bioactive chemical constituents from Alchornea laxiflora (benth)
pax and Hoffman. J Ethnopharm 74:275–280
3. Block S, Baccelli C, Tinant B, Meervelt LV, Rozenberg R, Jiwan
`
HJ-L, Llabres G, De Pauw-Gillet M-C, Quetin-Leclercq J (2004)
Diterpenes from the leaves of Croton zambesicus. Phytochemis-
try 65:1165–1171
4. Zhong-Jian J, Xiang-Qian L, Zi-Min L (1993) Triterpenoids from
Sanguisorba alpina. Phytochemistry 32:155–159
5. Sabira B, Ishrat S, Bina SS, Farhana S, Anwar HG (2002)
Structure and spasmolytic activity of eucalyptanoic acid from
Eucalyptus camaldulensis var. obtusa and synthesis of its active
derivative from oleanolic acid. J Nat Prod 65:1939–1941
6. Gabriela EF, Alejandro T, Angel GR, Carla D, Nadine BE,
Guillermo S-H (2002) Free radical scavengers, anti-inflammatory
and analgesic activity of Acaena magellanica. J Pharm Pharma-
col 54:835–844
7. Kim J-P, Lee I-K, Yun B-S, Chung S-H, Shim G-S, Hiroyuki K,
Yoo I-D (2001) Ellagic acid rhamnosides from the stem bark of
Eucalyptus globulus. Phytochemistry 57:587–591
8. Walter ED (1963) Isolation of b-sitosterol, b-sitosteryl-D-gluco-
side, and palmitic acid from Coastal Bermuda grass and Orchard
grass. J Pharm Sci 52:708
Conclusion
A. laxiflora has been investigated chemically and biologi-
cally and reports indicated that it contains several com-
ponents with antimicrobial and antioxidant activities
[2, 17] which are probably related to its ethnopharmaco-
logic uses [17, 18]. The work presented provides additional
characterization of this plant by isolating selected bark
components metabolites and that may help explain its wide
use in the Cameroonian traditional pharmacopeia. [19, 20]
123

J Am Oil Chem Soc (2011) 88:1153–1159
1159
9. Siddiqui S, Hafeez F, Begum S, Siddiqui BS (1988) Oleanderol, a
new pentacyclic triterpene from the leaves of Nerium oleander.
J Nat Prod 51:229–233
10. Guan Y, Hua P, Mingsong F, Huang C-G (2007) Ellagic acid
derivatives from the stem bark of Dipentodon sinicus. Chem Nat
Comp 43:125–127
16. Kishida E, Megumi K, Yumiko T, Matsumura M, Hayashi T,
Kobayashi S, Masuzawa Y (1997) Evaluation of a trans config-
uration for the apoptosis-inducing activity of ceramide. J Lipid
Mediat Cell Signal 16:127–137
17. Olatunde FE, Oluyemisi OO, Uhunwangho ES, Deyanju MA,
Olarenwaju MJ (2003) Antioxidant properties of extracts from
Alchornea laxiflora (Benth) Pax and Hoffman. Phytother Res
17:713–716
11. Yonghong L, Jee HJ, Hong J, Si Z (2006) Glycerolipids from a
Sarcotragus species sponge. Molecules 11:714–719
ˇ
12. Cateni F, Falsone G, Zilic J, Bonivento P, Zacchigna M, Zigon D,
18. Ganiyat KO, Akpomedaye OP, SOYINKA J, Olu WO, THONDA
E (2010) Phytochemical screening, antimicrobial and antioxidant
activities of four Nigerian medicinal plants. Ann Biol Res
1:114–120
19. Focho DA, Nkeng EAP, Lucha CF, Ndam WT, Afegenui A
(2009) Ethnobotanical survey of plants used to treat diseases of
the reproductive system and preliminary phytochemical screening
of some species of Malvaceae in Ndop Central Sub-division,
Cameroon. J Med Plants Res 3:301–314
Sosa S, Altinier G (2004) Glyceroglycolipids from Euphorbia
nicaeensis. All with antiinflammatory activity. Arkivoc v:54–65
13. Costautino V, Fattorusso E, Mangoni A (1993) An unusual ether
glycolipid from the Senegalese sponge Trikentrion loeve Carter.
Tetrahedron 49:2711–2716
14. Kim SY, Choi Y-H, Huh H, Kim J, Kim YC, Lee HS (1997) New
antihepatotoxic cerebroside from Lycium chinense fruits. J Nat
Prod 60:274–276
15. Cateni F, Zilic J, Falsone G, Scialino G, Banfi E (2003) New
cerebrosides from Euphorbia peplis L. Antimicrobial activity
evaluation. Bioorg Med Chem Lett 13:4345–4350
20. Jiofack T, Ayissi l, Fokunang C, Guedje N, Kemeuze V (2009)
Ethnobotany and phytomedicine of the upper Nyong valley forest
in Cameroon. Afr J Pharm Pharmacol 3:144–150
123